Methods for detecting vitamin C by mass spectrometry

ABSTRACT

Provided are methods for determining the amount of vitamin C in a sample using mass spectrometry. The methods generally involve ionizing vitamin C in a sample and detecting and quantifying the amount of the ion to determine the amount of vitamin C in the sample.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a Continuation of U.S. application Ser. No.13/078,792, filed Apr. 1, 2011, which is a continuation of U.S.application Ser. No. 12/269,862, filed Nov. 12, 2008 which claimsbenefit of U.S. Provisional Application No. 61/103,212, filed Oct. 6,2008, each of which is incorporated by reference herein in its entiretyincluding all figures and tables.

FIELD OF THE INVENTION

The invention relates to the detection of Vitamin C. In a particularaspect, the invention relates to methods for detecting Vitamin C by massspectrometry.

BACKGROUND OF THE INVENTION

The following description of the background of the invention is providedsimply as an aid in understanding the invention and is not admitted todescribe or constitute prior art to the invention.

Vitamin C [2-oxo-L-threo-hexono-1,4-lactone2,3-enediol] or L-ascorbicacid is a water-soluble vitamin and essential nutrient for humans. It isessential in the formation of collagen, which is required for normalgrowth and development as well as tissue repair in all parts of thebody. Vitamin C also functions as an antioxidant that blocks the damagecaused by free radicals and directly reduces toxic chemicals andpollutants.

As humans do not produce vitamin C in the body, it is primarily obtainedfrom dietary sources such as fruits and vegetables. Lack of dietaryvitamin C may result in vitamin C deficiency. Severe vitamin Cdeficiency, also know as “scurvy,” leads to the formation of liver spotson skin, spongy gums, and bleeding from mucous membranes, or even death.

Currently, vitamin C is not only used as a dietary supplement, but alsoas an adjunct therapy for some viral infections and terminal cancers.The recommended daily intake of vitamin C for adults to preventdeficiency is 75 mg for females and 90 mg for males, both with atolerable upper level of 2,000 mg. For therapeutic usage indetoxification and cancer therapy, vitamin C is given intravenously atmuch higher doses. Although vitamin C toxicity is rare clinically,relatively high doses of oral intake may lead to stomach upset anddiarrhea.

Assays for vitamin C blood levels have been developed and are used bypatients and physicians to evaluate nutritional status or to optimizetherapeutic dosages.

SUMMARY OF THE INVENTION

The present invention provides methods for detecting the amount ofvitamin C in a sample by mass spectrometry, including tandem massspectrometry.

In one aspect, methods are provided for determining the amount ofvitamin C in a test sample. Methods of this aspect include: (a) ionizingvitamin C from the test sample to produce one or more vitamin C ionsdetectable by mass spectrometry; and (b) detecting the amount of thevitamin C ion(s) by mass spectrometry. Once the amount of the one ormore vitamin C ions is measured, the amount of vitamin C ion(s) isrelated to the amount of vitamin C in the test sample. In these methods,the one or more vitamin C ions detectable by tandem mass spectrometryinclude one or more ions selected from the group consisting of ions witha mass/charge ratio of 175.05±0.5, 114.85±0.5, and 86.85±0.5. In someembodiments, the mass spectrometry is tandem mass spectrometry. In someembodiments, the methods further comprise purifying vitamin C in thetest samples prior to mass spectrometry. In related embodiments, saidpurifying comprises purifying by liquid chromatography. In furtherrelated embodiments, the liquid chromatography is high performanceliquid chromatography (HPLC). In some embodiments, purifying vitamin Ccomprises one or more purification steps prior to liquid chromatography.In related embodiments, the one or more purification steps precedingliquid chromatography may include protein precipitation. In someembodiments, the test sample is body fluid; preferably plasma or serum.In some embodiments, the step of ionizing vitamin C includes generatinga precursor ion with a mass/charge ratio of 175.05±0.5, and generatingone or more fragment ions selected from the group consisting of ionswith a mass/charge ratio of 114.85±0.5, and 86.85±0.5. In someembodiments, a stabilizing agent may be added to the test sample priorto mass spectrometry. In related embodiments, the stabilizing agent istrichloroacetic acid (TCA). In some embodiments, the method has a lowerlimit of quantitation within the range of 10.0 mg/dL and 0.1 mg/dL,inclusive. In some embodiments, the amount of one or more vitamin C ionsdetermined by mass spectrometry is related to the presence or amount ofvitamin C in the test sample by comparison to an internal standard;preferably ¹³C₆-L-ascorbic acid.

In a second aspect, methods are provided for determining the amount ofvitamin C in a body fluid sample by mass spectrometry. Methods of thisaspect include: (a) purifying vitamin C in a body fluid sample; (b)ionizing vitamin C from the body fluid sample to produce one or morevitamin C ions detectable by mass spectrometry; and (c) detecting theamount of the vitamin C ion(s) by mass spectrometry. In these methods,the amount of the vitamin C ion(s) determined by mass spectrometry isrelated to the amount of vitamin C in the test sample. In someembodiments, the mass spectrometry is tandem mass spectrometry. In someembodiments, the body fluid samples are purified by liquidchromatography. In related embodiments, the liquid chromatography may behigh performance liquid chromatography (HPLC). In some embodiments, thestep of purifying vitamin C in a test sample includes one or morepurification steps prior to liquid chromatography. In relatedembodiments, the one or more purification steps preceding liquidchromatography may include protein precipitation. In some embodiments,the test sample is plasma or serum. In some embodiments, the vitamin Cions detectable by mass spectrometry include one or more ions selectedfrom the group consisting of ions with a mass/charge ratio of175.05±0.5, 114.85±0.5, and 86.85±0.5. In some embodiments, the step ofionizing vitamin C includes generating a precursor ion with amass/charge ratio of 175.05±0.5, and generating one or more fragmentions selected from the group consisting of ions with a mass/charge ratioof 114.85±0.5, and 86.85±0.5. In some embodiments, a stabilizing agentmay be added to the test sample prior to mass spectrometry; preferablyprior to purifying the test sample. In related embodiments, thestabilizing agent is trichloroacetic acid (TCA). In some embodiments,the method has a lower limit of quantitation within the range of 10.0mg/dL and 0.1 mg/dL, inclusive. In some embodiments, the amount of oneor more vitamin C ions determined by mass spectrometry is related to thepresence or amount of vitamin C in the test sample by comparison to aninternal standard; preferably ¹³C₆-L-ascorbic acid.

In at third aspect, methods are provided for determining the amount ofvitamin C in a body fluid sample by tandem mass spectrometry. Methods ofthis aspect include: (a) purifying vitamin C from a body fluid sample byliquid chromatography; (b) generating a precursor ion of vitamin Chaving a mass/charge ratio of 175.05±0.5; (c) generating one or morefragment ions of the precursor ion selected from the group of fragmentions having a mass/charge ratio of 114.85±0.5, and 86.85±0.5; and (d)detecting the amount of one or more of the ions generated in step (b) or(c) or both and relating the determined ions to the amount of vitamin Cin the body fluid sample. In some embodiments, the method has a lowerlimit of quantitation within the range of 10.0 mg/dL and 0.1 mg/dL,inclusive. In some embodiments, liquid chromatography is highperformance liquid chromatography (HPLC). In some embodiments, the stepof purifying vitamin C in a body fluid sample includes one or morepurification steps prior to liquid chromatography. In relatedembodiments, the one or more purification steps preceding liquidchromatography may include protein precipitation. In some embodiments,the test sample is plasma or serum. In some embodiments, a stabilizingagent may be added to the test sample prior to mass spectrometry;preferably prior to purifying the test sample. In related embodiments,the stabilizing agent is trichloroacetic acid (TCA). In someembodiments, the method has a lower limit of quantitation within therange of 10.0 mg/dL and 0.1 mg/dL, inclusive. In some embodiments, theamount of one or more vitamin C ions determined by mass spectrometry isrelated to the presence or amount of vitamin C in the test sample bycomparison to an internal standard; preferably ¹³C₆-L-ascorbic acid.

Methods of the present invention involve the combination of liquidchromatography with mass spectrometry. In preferred embodiments, theliquid chromatography is HPLC. One preferred embodiment utilizes HPLCalone or in combination with one or more purification methods such asfor example HTLC or protein precipitation and filtration, to purifyvitamin C in samples. In another preferred embodiment, the massspectrometry is tandem mass spectrometry (MS/MS).

In certain preferred embodiments of the methods disclosed herein, massspectrometry is performed in negative ion mode. Alternatively, massspectrometry is performed in positive ion mode. Various ionizationsources, including for example atmospheric pressure chemical ionization(APCI) or electrospray ionization (ESI), may be used in embodiments ofthe present invention. In certain preferred embodiments, vitamin C ismeasured using APCI in negative mode.

In preferred embodiments, vitamin C ions detectable in a massspectrometer are selected from the group consisting of negative ionswith a mass/charge ratio (m/z) of 175.05±0.50, 114.85±0.50, and86.85±0.50. In particularly preferred embodiments, a vitamin C precursorion has m/z of 175.05±0.50, and one or more fragment ions are selectedfrom the group consisting of ions having m/z of 114.85±0.50 and86.85±0.50.

In preferred embodiments, a separately detectable internal vitamin Cstandard is provided in the sample, the amount of which is alsodetermined in the sample. In these embodiments, all or a portion of boththe endogenous vitamin C and the internal standard present in the sampleis ionized to produce a plurality of ions detectable in a massspectrometer, and one or more ions produced from each are detected bymass spectrometry.

A preferred internal vitamin C standard is ¹³C₆-L-ascorbic acid. Inpreferred embodiments, the internal vitamin C standard ions detectablein a mass spectrometer are selected from the group consisting ofnegative ions with m/z of 181.10±0.50, 119.10±0.50, and 90.00±0.50. Inparticularly preferred embodiments, a precursor ion of the internalvitamin C standard has m/z of 181.10±0.50; and one or more fragment ionsare selected from the group consisting of ions having m/z of119.10±0.50, and 90.00±0.50.

In preferred embodiments, the presence or amount of the vitamin C ion isrelated to the presence or amount of vitamin C in the test sample bycomparison to a reference such as ¹³C₆-L-ascorbic acid.

In certain preferred embodiments, the lower limit of quantitation (LLOQ)of vitamin C is within the range of 10.0 mg/dL and 0.1 mg/dL, inclusive;preferably within the range of 5.0 mg/dL and 0.1 mg/dL; preferablywithin the range of 2.5 mg/dL and 0.1 mg/dL; preferably within the rangeof 1.0 mg/dL and 0.1 mg/dL; preferably within the range of 0.50 mg/dLand 0.1 mg/dL; preferably within the range of 0.40 mg/dL and 0.1 mg/dL;preferably within the range of 0.30 mg/dL and 0.1 mg/dL; preferablywithin the range of 0.20 mg/dL and 0.1 mg/dL; preferably about 0.1mg/dL.

As used herein, unless otherwise stated, the singular forms “a,” “an,”and “the” include plural reference. Thus, for example, a reference to “aprotein” includes a plurality of protein molecules.

As used herein, the term “purification” or “purifying” does not refer toremoving all materials from the sample other than the analyte(s) ofinterest. Instead, purification refers to a procedure that enriches theamount of one or more analytes of interest relative to other componentsin the sample that may interfere with detection of the analyte ofinterest. Purification of the sample by various means may allow relativereduction of one or more interfering substances, e.g., one or moresubstances that may or may not interfere with the detection of selectedvitamin C parent or daughter ions by mass spectrometry. Relativereduction as this term is used does not require that any substance,present with the analyte of interest in the material to be purified, isentirely removed by purification.

As used herein, the term “test sample” refers to any sample that maycontain vitamin C. As used herein, the term “body fluid” means any fluidthat can be isolated from the body of an individual. For example, “bodyfluid” may include blood, plasma, serum, bile, saliva, urine, tears,perspiration, and the like.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas is separated into componentsas a result of differential distribution of the chemical entities asthey flow around or over a stationary liquid or solid phase.

As used herein, the term “liquid chromatography” or “LC” means a processof selective retardation of one or more components of a fluid solutionas the fluid uniformly percolates through a column of a finely dividedsubstance, or through capillary passageways. The retardation resultsfrom the distribution of the components of the mixture between one ormore stationary phases and the bulk fluid, (i.e., mobile phase), as thisfluid moves relative to the stationary phase(s). Examples of “liquidchromatography” include reverse phase liquid chromatography (RPLC), highperformance liquid chromatography (HPLC), and high turbulence liquidchromatography (HTLC).

As used herein, the term “high performance liquid chromatography” or“HPLC” refers to liquid chromatography in which the degree of separationis increased by forcing the mobile phase under pressure through astationary phase on a support matrix, typically a densely packed column.

As used herein, the term “high turbulence liquid chromatography” or“HTLC” refers to a form of chromatography that utilizes turbulent flowof the material being assayed through the column packing as the basisfor performing the separation. HTLC has been applied in the preparationof samples containing two unnamed drugs prior to analysis by massspectrometry. See, e.g., Zimmer et al., J. Chromatogr. A 854: 23-35(1999); see also, U.S. Pat. Nos. 5,968,367, 5,919,368, 5,795,469, and5,772,874, which further explain HTLC. Persons of ordinary skill in theart understand “turbulent flow”. When fluid flows slowly and smoothly,the flow is called “laminar flow”. For example, fluid moving through anHPLC column at low flow rates is laminar. In laminar flow the motion ofthe particles of fluid is orderly with particles moving generally instraight lines. At faster velocities, the inertia of the water overcomesfluid frictional forces and turbulent flow results. Fluid not in contactwith the irregular boundary “outruns” that which is slowed by frictionor deflected by an uneven surface. When a fluid is flowing turbulently,it flows in eddies and whirls (or vortices), with more “drag” than whenthe flow is laminar. Many references are available for assisting indetermining when fluid flow is laminar or turbulent (e.g., TurbulentFlow Analysis: Measurement and Prediction, P. S. Bernard & J. M.Wallace, John Wiley & Sons, Inc., (2000); An Introduction to TurbulentFlow, Jean Mathieu & Julian Scott, Cambridge University Press (2001)).

As used herein, the term “gas chromatography” or “GC” refers tochromatography in which the sample mixture is vaporized and injectedinto a stream of carrier gas (as nitrogen or helium) moving through acolumn containing a stationary phase composed of a liquid or aparticulate solid and is separated into its component compoundsaccording to the affinity of the compounds for the stationary phase.

As used herein, the term “large particle column” or “extraction column”refers to a chromatography column containing an average particlediameter greater than about 35 μm. As used in this context, the term“about” means±10%.

As used herein, the term “analytical column” refers to a chromatographycolumn having sufficient chromatographic plates to effect a separationof materials in a sample that elute from the column sufficient to allowa determination of the presence or amount of an analyte. Such columnsare often distinguished from “extraction columns”, which have thegeneral purpose of separating or extracting retained material fromnon-retained materials in order to obtain a purified sample for furtheranalysis. As used in this context, the term “about” means±10%. In apreferred embodiment the analytical column contains particles of about3.5 μm in diameter.

As used herein, the term “on-line” or “inline”, for example as used in“on-line automated fashion” or “on-line extraction” refers to aprocedure performed without the need for operator intervention. Incontrast, the term “off-line” as used herein refers to a procedurerequiring manual intervention of an operator. Thus, if samples aresubjected to precipitation, and the supernatants are then manuallyloaded into an autosampler, the precipitation and loading steps areoff-line from the subsequent steps. In various embodiments of themethods, one or more steps may be performed in an on-line automatedfashion.

As used herein, the term “mass spectrometry” or “MS” refers to ananalytical technique to identify compounds by their mass. MS refers tomethods of filtering, detecting, and measuring ions based on theirmass-to-charge ratio, or “m/z”. MS technology generally includes (1)ionizing the compounds to form charged compounds; and (2) detecting themolecular weight of the charged compounds and calculating amass-to-charge ratio. The compounds may be ionized and detected by anysuitable means. A “mass spectrometer” generally includes an ionizer andan ion detector. In general, one or more molecules of interest areionized, and the ions are subsequently introduced into a massspectrographic instrument where, due to a combination of magnetic andelectric fields, the ions follow a path in space that is dependent uponmass (“m”) and charge (“z”). See, e.g., U.S. Pat. Nos. 6,204,500,entitled “Mass Spectrometry From Surfaces;” 6,107,623, entitled “Methodsand Apparatus for Tandem Mass Spectrometry;” 6,268,144, entitled “DNADiagnostics Based On Mass Spectrometry;” 6,124,137, entitled“Surface-Enhanced Photolabile Attachment And Release For Desorption AndDetection Of Analytes;” Wright et al., Prostate Cancer and ProstaticDiseases 2:264-76 (1999); and Merchant and Weinberger, Electrophoresis21:1164-67 (2000).

As used herein, the term “operating in negative ion mode” refers tothose mass spectrometry methods where negative ions are generated anddetected. The term “operating in positive ion mode” as used herein,refers to those mass spectrometry methods where positive ions aregenerated and detected.

As used herein, the term “ionization” or “ionizing” refers to theprocess of generating an analyte ion having a net electrical chargeequal to one or more electron units. Negative ions are those having anet negative charge of one or more electron units, while positive ionsare those having a net positive charge of one or more electron units.

As used herein, the term “electron ionization” or “EI” refers to methodsin which an analyte of interest in a gaseous or vapor phase interactswith a flow of electrons. Impact of the electrons with the analyteproduces analyte ions, which may then be subjected to a massspectrometry technique.

As used herein, the term “chemical ionization” or “CI” refers to methodsin which a reagent gas (e.g. ammonia) is subjected to electron impact,and analyte ions are formed by the interaction of reagent gas ions andanalyte molecules.

As used herein, the term “fast atom bombardment” or “FAB” refers tomethods in which a beam of high energy atoms (often Xe or Ar) impacts anon-volatile sample, desorbing and ionizing molecules contained in thesample. Test samples are dissolved in a viscous liquid matrix such asglycerol, thioglycerol, m-nitrobenzyl alcohol, 18-crown-6 crown ether,2-nitrophenyloctyl ether, sulfolane, diethanolamine, andtriethanolamine. The choice of an appropriate matrix for a compound orsample is an empirical process.

As used herein, the term “matrix-assisted laser desorption ionization”or “MALDI” refers to methods in which a non-volatile sample is exposedto laser irradiation, which desorbs and ionizes analytes in the sampleby various ionization pathways, including photo-ionization, protonation,deprotonation, and cluster decay. For MALDI, the sample is mixed with anenergy-absorbing matrix, which facilitates desorption of analytemolecules.

As used herein, the term “surface enhanced laser desorption ionization”or “SELDI” refers to another method in which a non-volatile sample isexposed to laser irradiation, which desorbs and ionizes analytes in thesample by various ionization pathways, including photo-ionization,protonation, deprotonation, and cluster decay. For SELDI, the sample istypically bound to a surface that preferentially retains one or moreanalytes of interest. As in MALDI, this process may also employ anenergy-absorbing material to facilitate ionization.

As used herein, the term “electrospray ionization” or “ESI,” refers tomethods in which a solution is passed along a short length of capillarytube, to the end of which is applied a high positive or negativeelectric potential. Solution reaching the end of the tube is vaporized(nebulized) into a jet or spray of very small droplets of solution insolvent vapor. This mist of droplets flows through an evaporationchamber. As the droplets get smaller the electrical surface chargedensity increases until such time that the natural repulsion betweenlike charges causes ions as well as neutral molecules to be released.

As used herein, the term “atmospheric pressure chemical ionization” or“APCI,” refers to mass spectrometry methods that are similar to ESI;however, APCI produces ions by ion-molecule reactions that occur withina plasma at atmospheric pressure. The plasma is maintained by anelectric discharge between the spray capillary and a counter electrode.Then ions are typically extracted into the mass analyzer by use of a setof differentially pumped skimmer stages. A counterflow of dry andpreheated N₂ gas may be used to improve removal of solvent. Thegas-phase ionization in APCI can be more effective than ESI foranalyzing less-polar species.

The term “atmospheric pressure photoionization” or “APPI” as used hereinrefers to the form of mass spectrometry where the mechanism for thephotoionization of molecule M is photon absorption and electron ejectionto form the molecular ion M+. Because the photon energy typically isjust above the ionization potential, the molecular ion is lesssusceptible to dissociation. In many cases it may be possible to analyzesamples without the need for chromatography, thus saving significanttime and expense. In the presence of water vapor or protic solvents, themolecular ion can extract H to form MH+. This tends to occur if M has ahigh proton affinity. This does not affect quantitation accuracy becausethe sum of M+ and MH+ is constant. Drug compounds in protic solvents areusually observed as MH+, whereas nonpolar compounds such as naphthaleneor testosterone usually form M+. Robb, D. B., Covey, T. R. and Bruins,A. P. (2000): See, e.g., Robb et al., Atmospheric pressurephotoionization: An ionization method for liquid chromatography-massspectrometry. Anal. Chem. 72(15): 3653-3659.

As used herein, the term “inductively coupled plasma” or “ICP” refers tomethods in which a sample interacts with a partially ionized gas at asufficiently high temperature such that most elements are atomized andionized.

As used herein, the term “field desorption” refers to methods in which anon-volatile test sample is placed on an ionization surface, and anintense electric field is used to generate analyte ions.

As used herein, the term “desorption” refers to the removal of ananalyte from a surface and/or the entry of an analyte into a gaseousphase.

As used herein, the term “selective ion monitoring” is a detection modefor a mass spectrometric instrument in which only ions within arelatively narrow mass range, typically about one mass unit, aredetected.

As used herein, “multiple reaction mode,” sometimes known as “selectedreaction monitoring,” is a detection mode for a mass spectrometricinstrument in which a precursor ion and one or more fragment ions areselectively detected.

As used herein, the term “limit of quantification”, “limit ofquantitation” or “LOQ” refers to the point where measurements becomequantitatively meaningful. The analyte response at this LOQ isidentifiable, discrete and reproducible with a relative standarddeviation (RSD %) of 20% and an accuracy of 85% to 115%.

As used herein, the term “limit of detection” or “LOD” is the point atwhich the measured value is larger than the uncertainty associated withit. The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the LOQ.

As used herein, an “amount” of vitamin C in a body fluid sample refersgenerally to an absolute value reflecting the mass of vitamin Cdetectable in volume of body fluid. However, an amount also contemplatesa relative amount in comparison to another vitamin C amount. Forexample, an amount of vitamin C in a body fluid can be an amount whichis greater than a control or normal level of vitamin C normally present.

The term “about” as used herein in reference to quantitativemeasurements not including the measurement of the mass of an ion, refersto the indicated value plus or minus 10%. Mass spectrometry instrumentscan vary slightly in determining the mass of a given analyte. The term“about” in the context of the mass of an ion or the mass/charge ratio ofan ion refers to +/−0.50 atomic mass unit.

The summary of the invention described above is non-limiting and otherfeatures and advantages of the invention will be apparent from thefollowing detailed description of the invention, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows exemplary chromatograms of vitamin C and ¹³C₆-L-ascorbicacid (internal standard). Details are discussed in Example 3.

FIG. 2 shows the linearity of the quantitation of vitamin C in seriallydiluted stock samples using an LC-MS/MS assay. Details are described inExample 6.

DETAILED DESCRIPTION OF THE INVENTION

Methods for detecting vitamin C using liquid chromatography coupled withvarious detection means have been described in the art. For example TaiA, et al., J Chromatogr B 2006, 840:38-43; Zafra-Gómez A, et al., JAgric Food Chem 2006, 54:4531-6; Walker, P, et al., Phytochem Anal 2006,17:284-90; Karlsen A, et al., Eur J Clin Nutr 2007, 61:1233-6; andLundegårdh B, et al., J Agric Food Chem 2008, 56:2102-11 describedetection of vitamin C using high performance liquid chromatography andultraviolet/visible light (UV-Vis) absorbance. Methods to detect vitaminC in food matrices by liquid chromatography/coulometric detection aredisclosed in Franke, A., et al., J Food Comp Anal. 2004, 17:1-35.Methods to detect vitamin C in plasma by high performance liquidchromatography/electrochemical detection are disclosed in Salminen, I.,et al., Clin Biochem 2008, 41:723-7. A method to detect vitamin C infood matrices with high performance liquid chromatography/tandem massspectrometry by observing a mass transition from a precursor ion with amass to charge ratio of 177 to fragment ions with mass to charge ratiosof 141 and 95 is disclosed in Gentili, et al., Rapid Commun MassSpectrom 2008, 22:2029-43.

Methods of the present invention are described for measuring the amountof vitamin C in a sample. More specifically, mass spectrometric methodsare described for detecting and quantifying vitamin C in a test sample.The methods may utilize liquid chromatography (LC), most preferablyHPLC, to perform a purification of selected analytes, and combine thispurification with unique methods of mass spectrometry (MS), therebyproviding a high-throughput assay system for detecting and quantifyingvitamin C in a test sample. The preferred embodiments are particularlywell suited for application in large clinical laboratories for automatedvitamin C assay. The methods provided are accomplished with decreasedvitamin C degradation through sample treatment and preparation.

Suitable test samples for use in methods of the present inventioninclude any test sample that may contain the analyte of interest. Insome preferred embodiments, a sample is a biological sample; that is, asample obtained from any biological source, such as an animal, a cellculture, an organ culture, etc. In certain preferred embodiments,samples are obtained from a mammalian animal, such as a dog, cat, horse,etc. Particularly preferred mammalian animals are primates, mostpreferably male or female humans. Particularly preferred samples includebodily fluids such as blood, plasma, serum, saliva, cerebrospinal fluid,or a tissue sample. Such samples may be obtained, for example, from apatient; that is, a living person, male or female, presenting oneself ina clinical setting for diagnosis, prognosis, or treatment of a diseaseor condition. The test sample is preferably obtained from a patient, forexample, blood serum or plasma. Vitamin C in blood serum or plasma isvery temperature and light sensitive, so in order to avoid irreversibledegradation, samples should be protected from light and placed under dryice or ultra-low refrigeration and quickly thawed just prior to use. Asample volume of about 2 mL is preferred; however, samples of about 1 mLcan be analyzed.

In some embodiments of the present invention, test samples may also betreated prior to mass spectrometry with a stabilizing reagent to slowphoto and thermal degradation; preferably prior to purification. Inespecially preferred embodiments, the stabilizing reagent is thewell-known trichloroacetic acid (TCA). See for example, Bradley, et al.,Clinica Chemica Acta 1973, 44:47-52. Treatment of test samples with astabilizing reagent is especially useful in preparing multiple testsamples for automated analysis. Multiple test samples, for example 96samples, each in a well of a 96-well plate, can be prepared forautomated analysis by using a stabilizing reagent.

The present invention contemplates kits for an vitamin C quantitationassay. A kit for an vitamin C quantitation assay of the presentinvention may include a kit comprising trichloroacetic acid (TCA) and aninternal standard, in amounts sufficient for at least one assay.Typically, the kits will also include instructions recorded in atangible form (e.g., contained on paper or an electronic medium) forusing the packaged reagents for use in a measurement assay fordetermining the amount of vitamin C.

Calibration and QC pools for use in embodiments of the present inventioncan be prepared using “stripped” plasma or serum (stripped of vitaminC): for example, analyte-stripped, defibrinated and delipidizedplasma/serum. All sources of human or non-human plasma or stripped serumshould be checked to ensure that they do not contain measurable amountsof vitamin C.

Sample Preparation for Mass Spectrometry

Typically, frozen test samples (including controls) are thawed rapidlyand kept protected from light exposure to minimize vitamin Cdegradation. Internal standard may be added to the test samples oncethey are thawed.

The samples may then be prepared for mass spectrometry by liquid-liquidor solid-phase extraction, and/or treatment with a stabilizing reagent.Various methods may be used to enrich vitamin C relative to othercomponents in the sample (e.g. protein) prior mass spectrometry,including for example, liquid chromatography, filtration,centrifugation, thin layer chromatography (TLC), electrophoresisincluding capillary electrophoresis, affinity separations includingimmunoaffinity separations, extraction methods including ethyl acetateextraction and methanol extraction, and the use of chaotropic agents orany combination of the above or the like.

Protein precipitation is one preferred method of preparing a testsample, especially a biological test sample, such as serum or plasma.Such protein purification methods are well known in the art, forexample, Polson et al., Journal of Chromatography B 785:263-275 (2003),describes protein precipitation techniques suitable for use in methodsof the present invention. Protein precipitation may be used to removemost of the protein from the sample leaving vitamin C in thesupernatant. The samples may be centrifuged to separate the liquidsupernatant from the precipitated proteins; alternatively the samplesmay be filtered, for example through a glass fiber filter, to removeprecipitated proteins. The resultant supernatant or filtrate may then beapplied directly to mass spectrometry analysis; or alternatively toliquid chromatography and subsequent mass spectrometry analysis. Incertain embodiments, the use of protein precipitation such as forexample, methanol protein precipitation, may obviate the need for highturbulence liquid chromatography (HTLC) or other on-line extractionprior to mass spectrometry or HPLC and mass spectrometry.

Accordingly, in some embodiments, the method involves (1) performing aprotein precipitation of the sample of interest; and (2) loading thesupernatant directly onto the LC-mass spectrometer without using on-lineextraction or high turbulence liquid chromatography (HTLC).

In some embodiments, HTLC, alone or in combination with one or morepurification methods, may be used to purify vitamin C prior to massspectrometry. In such embodiments samples may be extracted using an HTLCextraction cartridge which captures the analyte, then eluted andchromatographed on a second HTLC column or onto an analytical HPLCcolumn prior to ionization. Because the steps involved in thesechromatography procedures may be linked in an automated fashion, therequirement for operator involvement during the purification of theanalyte can be minimized. This feature may result in savings of time andcosts, and eliminate the opportunity for operator error.

According to preferred embodiments, the method involves adding astabilizing reagent, such as for example trichloroacetic acid (TCA), toeach sample prior to mass spectrometry, for example prior to one or morepurification steps, for example prior to storage. In some embodiments,TCA may be added to each sample after protein precipitation, but priorto liquid chromatography.

One means of sample purification that may be used prior to massspectrometry is liquid chromatography (LC). Liquid chromatography,including high-performance liquid chromatography (HPLC), relies onrelatively slow, laminar flow technology. Traditional HPLC analysisrelies on column packing in which laminar flow of the sample through thecolumn is the basis for separation of the analyte of interest from thesample. The skilled artisan will understand that separation in suchcolumns is a diffusional process and may select HPLC instruments andcolumns that are suitable for use with vitamin C. The chromatographiccolumn typically includes a medium (i.e., a packing material) tofacilitate separation of chemical moieties (i.e., fractionation). Themedium may include minute particles. The particles include a bondedsurface that interacts with the various chemical moieties to facilitateseparation of the chemical moieties. One suitable bonded surface is ahydrophobic bonded surface such as an alkyl bonded surface. Alkyl bondedsurfaces may include C-4, C-8, C-12, or C-18 bonded alkyl groups,preferably C-18 bonded groups. The chromatographic column includes aninlet port for receiving a sample directly or indirectly from coupledSPE column and an outlet port for discharging an effluent that includesthe fractionated sample.

In one embodiment, the sample may be applied to the column at the inletport, eluted with a solvent or solvent mixture, and discharged at theoutlet port. Different solvent modes may be selected for eluting theanalyte(s) of interest. For example, liquid chromatography may beperformed using a gradient mode, an isocratic mode, or a polytyptic(i.e. mixed) mode. During chromatography, the separation of materials iseffected by variables such as choice of eluent (also known as a “mobilephase”), elution mode, gradient conditions, temperature, etc.

In certain embodiments, an analyte may be purified by applying a sampleto a column under conditions where the analyte of interest is reversiblyretained by the column packing material, while one or more othermaterials are not retained. In these embodiments, a first mobile phasecondition can be employed where the analyte of interest is retained bythe column, and a second mobile phase condition can subsequently beemployed to remove retained material from the column, once thenon-retained materials are washed through. Alternatively, an analyte maybe purified by applying a sample to a column under mobile phaseconditions where the analyte of interest elutes at a differential ratein comparison to one or more other materials. Such procedures may enrichthe amount of one or more analytes of interest relative to one or moreother components of the sample.

In one preferred embodiment, HPLC is conducted with a hydrophobic columnchromatographic system. In certain preferred embodiments, a C18analytical column (e.g., a Zorbax SB-C18 analytical column from AgilentTechnologies (3.5 μm particle size, 15×4.6 mm), or equivalent) is used.In certain preferred embodiments, HTLC and/or HPLC are performed usingHPLC Grade 0.1% aqueous formic acid and 100% acetonitrile as the mobilephases.

By careful selection of valves and connector plumbing, two or morechromatography columns may be connected as needed such that material ispassed from one to the next without the need for any manual steps. Inpreferred embodiments, the selection of valves and plumbing iscontrolled by a computer pre-programmed to perform the necessary steps.Most preferably, the chromatography system is also connected in such anon-line fashion to the detector system, e.g., an MS system. Thus, anoperator may place a tray of samples in an autosampler, and theremaining operations are performed under computer control, resulting inpurification and analysis of all samples selected.

Detection and Quantitation by Mass Spectrometry

In various embodiments, vitamin C present in a test sample may beionized by any method known to the skilled artisan. Mass spectrometry isperformed using a mass spectrometer, which includes an ion source forionizing the fractionated sample and creating charged molecules forfurther analysis. For example ionization of the sample may be performedby electron ionization, chemical ionization, electrospray ionization(ESI), photon ionization, atmospheric pressure chemical ionization(APCI), photoionization, atmospheric pressure photoionization (APPI),fast atom bombardment (FAB), liquid secondary ionization (LSI), matrixassisted laser desorption ionization (MALDI), field ionization, fielddesorption, thermospray/plasmaspray ionization, surface enhanced laserdesorption ionization (SELDI), inductively coupled plasma (ICP) andparticle beam ionization. The skilled artisan will understand that thechoice of ionization method may be determined based on the analyte to bemeasured, type of sample, the type of detector, the choice of positiveversus negative mode, etc.

In preferred embodiments, vitamin C is ionized by atmospheric pressurechemical ionization (APCI) in negative mode. In related preferredembodiments, vitamin C ion is in a gaseous state and the inert collisiongas is argon or nitrogen; preferably argon.

In mass spectrometry techniques generally, after the sample has beenionized the positively charged or negatively charged ions therebycreated may be analyzed to determine a mass-to-charge ratio. Suitableanalyzers for determining mass-to-charge ratios include quadrupoleanalyzers, ion traps analyzers, magnetic and electric sector analyzers,and time-of-flight analyzers. The ions may be detected using severaldetection modes. For example, selected ions may be detected, i.e. usinga selective ion monitoring mode (SIM), or alternatively, ions may bedetected using a scanning mode, e.g., multiple reaction monitoring (MRM)or selected reaction monitoring (SRM). Preferably, the mass-to-chargeratio is determined using a quadrupole analyzer. For example, in a“quadrupole” or “quadrupole ion trap” instrument, ions in an oscillatingradio frequency field experience a force proportional to the DCpotential applied between electrodes, the amplitude of the RF signal,and the mass/charge ratio. The voltage and amplitude may be selected sothat only ions having a particular mass/charge ratio travel the lengthof the quadrupole, while all other ions are deflected. Thus, quadrupoleinstruments may act as both a “mass filter” and as a “mass detector” forthe ions injected into the instrument.

One may enhance the resolution of the MS technique by employing “tandemmass spectrometry,” or “MS/MS”. In this technique, a precursor ion (alsocalled a parent ion) generated from a molecule of interest can befiltered in an MS instrument, and the precursor ion is subsequentlyfragmented to yield one or more fragment ions (also called daughter ionsor product ions) that are then analyzed in a second MS procedure. Bycareful selection of precursor ions, only ions produced by certainanalytes are passed to the fragmentation chamber, where collisions withatoms of an inert gas produce the fragment ions. Because both theprecursor and fragment ions are produced in a reproducible fashion undera given set of ionization/fragmentation conditions, the MS/MS techniquemay provide an extremely powerful analytical tool. For example, thecombination of filtration/fragmentation may be used to eliminateinterfering substances, and may be particularly useful in complexsamples, such as biological samples.

The mass spectrometer typically provides the user with an ion scan; thatis, the relative abundance of each ion with a particular mass/chargeover a given range (e.g., 100 to 1000 amu). The results of an analyteassay, that is, a mass spectrum, may be related to the amount of theanalyte in the original sample by numerous methods known in the art. Forexample, given that sampling and analysis parameters are carefullycontrolled, the relative abundance of a given ion may be compared to atable that converts that relative abundance to an absolute amount of theoriginal molecule. Alternatively, standards may be run with the samples,and a standard curve constructed based on ions generated from thosestandards. Using such a standard curve, the relative abundance of agiven ion may be converted into an absolute amount of the originalmolecule. In certain preferred embodiments, an internal standard is usedto generate a standard curve for calculating the quantity of vitamin C.Methods of generating and using such standard curves are well known inthe art and one of ordinary skill is capable of selecting an appropriateinternal standard. For example, an isotopically labeled vitamin C may beused as an internal standard; in certain preferred embodiments thestandard is ¹³C₆-L-ascorbic acid. Numerous other methods for relatingthe amount of an ion to the amount of the original molecule will be wellknown to those of ordinary skill in the art.

One or more steps of the methods may be performed using automatedmachines. In certain embodiments, one or more purification steps areperformed on-line, and more preferably all of the purification and massspectrometry steps may be performed in an on-line fashion.

In certain embodiments, such as MS/MS, where precursor ions are isolatedfor further fragmentation, collision activation dissociation is oftenused to generate the fragment ions for further detection. In CAD,precursor ions gain energy through collisions with an inert gas, andsubsequently fragment by a process referred to as “unimoleculardecomposition.” Sufficient energy must be deposited in the precursor ionso that certain bonds within the ion can be broken due to increasedvibrational energy.

In particularly preferred embodiments, vitamin C is detected and/orquantified using MS/MS as follows. The samples are subjected to liquidchromatography, preferably HPLC; the flow of liquid solvent from thechromatographic column enters the heated nebulizer interface of an MS/MSanalyzer; and the solvent/analyte mixture is converted to vapor in theheated tubing of the interface. The analyte (e.g., vitamin C), containedin the nebulized solvent, is ionized by the corona discharge needle ofthe interface, which applies a large voltage to the nebulizedsolvent/analyte mixture. The ions, e.g. precursor ions, pass through theorifice of the instrument and enter the first quadrupole. Quadrupoles 1and 3 (Q1 and Q3) are mass filters, allowing selection of ions (i.e.,selection of “precursor” and “fragment” ions in Q1 and Q3, respectively)based on their mass to charge ratio (m/z). Quadrupole 2 (Q2) is thecollision cell, where ions are fragmented. The first quadrupole of themass spectrometer (Q1) selects for molecules with the mass to chargeratios of vitamin C. Precursor ions with the correct mass/charge ratiosare allowed to pass into the collision chamber (Q2), while unwanted ionswith any other mass/charge ratio collide with the sides of thequadrupole and are eliminated. Precursor ions entering Q2 collide withneutral argon gas molecules and fragment. This process is calledcollision activated dissociation (CAD). The fragment ions generated arepassed into quadrupole 3 (Q3), where the fragment ions of vitamin C areselected while other ions are eliminated.

The methods may involve MS/MS performed in either positive or negativeion mode; preferably negative ion mode. Using standard methods wellknown in the art, one of ordinary skill is capable of identifying one ormore fragment ions of a particular precursor ion of vitamin C that maybe used for selection in quadrupole 3 (Q3).

As ions collide with the detector they produce a pulse of electrons thatare converted to a digital signal. The acquired data is relayed to acomputer, which plots counts of the ions collected versus time. Theresulting mass chromatograms are similar to chromatograms generated intraditional HPLC methods. The areas under the peaks corresponding toparticular ions, or the amplitude of such peaks, are measured and thearea or amplitude is correlated to the amount of the analyte ofinterest. In certain embodiments, the area under the curves, oramplitude of the peaks, for fragment ion(s) and/or precursor ions aremeasured to determine the amount of vitamin C. As described above, therelative abundance of a given ion may be converted into an absoluteamount of the original analyte, e.g., vitamin C, using calibrationstandard curves based on peaks of one or more ions of an internalmolecular standard, such as ¹³C₆-L-ascorbic acid.

The following examples serve to illustrate the invention. These examplesare in no way intended to limit the scope of the methods.

EXAMPLES Example 1 Sample (Plasma and Serum) and Reagent Preparation

Plasma samples were prepared by collecting blood in a light-protectedVacutainer tube with sodium heparin while refrigerated to about 2° C. to8° C. Samples were then centrifuged (about 2200-2500 rpm, about 800-1000g) for about 8 to 10 minutes while refrigerated to about 2° C. to 8° C.The resulting plasma was then transferred to dark-brown polypropylene orpolyethylene transport tubes to protect the samples from light. Thesamples were then placed under dry ice or ultra low freezer (i.e.,cooled to a temperature of about −65° C. to −75° C.) to protect fromdegradation. Samples that were not protected from light and samples thatwere shipped and/or stored at refrigerated (i.e., about 2° C. to 8° C.)or ambient temperatures were not used for analysis. Additionally,samples that exhibited gross hemolysis and/or lipemia were alsoexcluded.

Serum samples were prepared by collecting blood in a light-protectedVacutainer tube with no additives and allowed to clot for 20 to 30minutes while refrigerated to about 2° C. to 8° C. The samples were thencentrifuged (about 2200-2500 rpm, about 800-1000 g) for about 8 to 10minutes while refrigerated to about 2° C. to 8° C. The resulting serumwas then transferred as above for plasma.

Two vitamin C stock solutions were prepared. A vitamin C stock solutionfor standards of 100 mg/mL in 1% meta-phosphoric acid was prepared in anamber volumetric flask. A vitamin C stock solution for controls of 10mg/mL in 1% meta-phosphoric acid was prepared in an amber volumetricflask. Aliquots of stock solutions were protected from light and kept atabout −65° C. to −75° C.

¹³C₆-L-ascorbic acid (Quote #184286, MDX, Cerritos, Calif. 90703, orequivalent) was used to prepare a 1.0 mg/mL in methanol ¹³C₆-L-ascorbicacid internal standard stock solution, which was used to prepare a 10mcg/mL internal standard working solution: 1 mL of the ¹³C₆-L-ascorbicacid internal standard stock solution was diluted to volume with DIwater in a 100 mL volumetric flask.

Example 2 Extraction of Vitamin C from Plasma and Serum Using LiquidChromatography

Liquid chromatography (LC) samples were prepared by thawing standards,controls, and patient samples to room temperature. Standards, controls,and patient samples were then vortexed for about 5 to 10 seconds.

0.20 mL of each vortexed standard, control, and patient sample was thencombined with 1.0 mL of internal standard working solution (10 mcg/mL)and 0.20 mL of 50% aqueous methanol. These mixtures were centrifugallyfiltered (about 3000-3300 rpm) through a glass fiber filter for about 8to 10 minutes while refrigerated to about 2° C. to 8° C. 50 μL of 20%aqueous TCA was then added to the each filtrate. Adding the TCA at thispoint in the procedure helps stabilize vitamin C in the samples. Thefiltrate/TCA mixtures were then vortexed for about 20 to 25 seconds andpoured into the wells of a deep 96-well plate for insertion into anautosampler cooling unit.

Sample injection was performed with a Cohesive Technologies Aria TLX-1HTLC system operating in laminar flow mode using Aria OS V 1.5 or newersoftware. Acetonitrile and DI water were used as autosampler washsolutions.

The HTLC system automatically injected 10±4 μL of the above preparedsamples into the analytical column (Zorbax SB-C18, 15×4.6 mm, 3.5 μmcolumn). A binary HPLC gradient was applied to the analytical column, toseparate vitamin C from other analytes contained in the sample. Mobilephase A was 0.1% aqueous formic acid and mobile phase B was 100%acetonitrile. The HPLC gradient started with no organic mobile phase,ramped to 75% organic mobile phase at approximately 20 seconds, and to95% at approximately 45 seconds. The analytes eluted off the HPLC columnat approximately 30 seconds. The separated sample was then subjected toMS/MS for quantitation of vitamin C.

To determine interference from other vitamins or related compounds,blank sera was spiked with: 10 mcg/mL each of pyridoxine, pyridoxamine,pyridoxal, 4-pyridoxic acid, pyridoxal-5-phosphate, retinol, β-carotene,α-tocopherol, γ-tocopherol, riboflavin, riboflavin-5-phosphate, flavinadenine dinucleotide (FAD), thiamin, thiamin-5-phosphate,thiamin-pyrophosphate, pyrithiamin, folic acid, vitamin B12,1,6-¹³C-ascorbic acid, D-isoascorbic acid, vitamin D₂, and vitamin D₃; 1mcg/mL each of 1,25-OH vitamin D₂ and 1,25-OH vitamin D₃; and 40 mcg/mLeach of 25-OH vitamin D₂ and 25-vitamin D₃. To determine interferencefrom various drugs, blank sera was spiked with 10 mcg/mL each ofimipramine, desipramine, amitriptyline, nortriptyline, doxepin,desmethyl doxepin, flouxetine, norflouxetine, maprotiline, clomipramine,desmethyl clomipramine, mycophenolic acid, mycophenolic acidglucuronide, flexamide, amiodarone, desethylamiodarone, hydroxzine,propafenone, lamotrigine, gabapentin, zonisamide, and lidocain. Thesamples were subject to LC. Of these molecules, the only two thatco-eluted with vitamin C were D-isoascorbic acid and 1,6-¹³C-ascorbicacid, but they are synthetic compounds that do not naturally appear inthe human body. Further, vitamin C (L-ascorbic acid) and its D-isomerare not inter-convertible even at very high concentrations of 500 mg/dL.

Example 3 Detection and Quantitation of Vitamin C by MS/MS

MS/MS was performed using a Finnigan TSQ Quantum Ultra MS/MS system(Thermo Electron Corporation). The following software programs all fromThermoElectron were used in the Examples described herein: Quantum TuneMaster V 1.2 or newer, Xcalibur V 1.4 SR1 or newer, TSQ Quantum 1.4 ornewer, and LCQuan V 2.0 with SP1 or newer. Liquid solvent/analyteexiting the analytical HPLC column flowed to the heated nebulizerinterface of a Thermo Finnigan MS/MS analyzer. The solvent/analytemixture was converted to vapor in the heated tubing of the interface.Analytes in the nebulized solvent were ionized by APCI.

Ions passed to the first quadrupole (Q1), which selected ions with amass to charge ratio of 175.05±0.50 m/z. Ions entering Quadrupole 2 (Q2)collided with argon gas to generate ion fragments, which were passed toquadrupole 3 (Q3) for further selection. Simultaneously, the sameprocess using isotope dilution mass spectrometry was carried out with aninternal standard, ¹³C₆-L-ascorbic acid. The following mass transitionswere used for detection and quantitation during validation on negativepolarity.

TABLE 1 Mass Transitions for Vitamin C (Negative Polarity) AnalytePrecursor Ion (m/z) Product Ion (m/z) Vitamin C 175.05 86.85 and 114.85¹³C₆-L-ascorbic acid 181.10 90.00 and 119.10 (internal standard)

Exemplary chromatograms for vitamin C and ¹³C₆-L-ascorbic acid (internalstandard) are found in FIG. 1.

Example 4 Intra-Assay and Inter-Assay Precision and Accuracy

Three quality control (QC) pools were prepared from analyte-stripped,defibrinated, and delipidized serum, spiked with vitamin C to aconcentration of 0.4, 1.2, and 4.0 dg/mL.

Twenty aliquots from each of the three QC pools were analyzed in asingle assay to determine the reproducibility (RSD (%)) of a samplewithin an assay. The following values were determined:

TABLE 2 Intra-Assay Variation and Accuracy Level I Level II Level III(0.4 mg/dL) (1.2 mg/dL) (4.0 mg/dL) Mean 0.37 1.21 4.04 StandardDeviation 0.03 0.07 0.15 RSD (%) 7.2% 6.0% 3.6% Accuracy (%) 93.1%100.9% 101.0%

Ten aliquots from each of the three QC pools were assayed over five daysto determine the reproducibility (RSD %) between assays. The followingvalues were determined:

TABLE 3 Inter-Assay Variation and Accuracy Level I Level II Level III(0.4 mg/dL) (1.2 mg/dL) (4.0 mg/dL) Mean 0.38 1.20 4.01 StandardDeviation 0.04 0.11 0.36 RSD (%) 9.6% 9.5% 8.9% Accuracy (%) 94.1%100.0% 100.2%

Example 5 Analytical Sensitivity: Limit of Detection (LOD) and LowerLimit of Quantitation (LLOQ)

The LLOQ is the point where measurements become quantitativelymeaningful. The analyte response at this LOQ is identifiable, discreteand reproducible with a relative standard deviation (RSD) of 20% and anaccuracy of 85% to 115%. The LLOQ was determined by assaying plasmaspecimens spiked with vitamin C concentrations of 2.50, 1.00, 0.50,0.25, 0.10, and 0.05 mg/dL (ten replicates each for five days at eachlevel) then determining the reproducibility. The LOQ for the vitamin Cassay was determined to be 0.1 mg/dL

The LOD is the point at which a value is beyond the uncertaintyassociated with its measurement and is defined as three times the RSD ofthe mean at the LLOQ. To determine the LOQ for the vitamin C assay, the0.10 mcg/mL vitamin C in plasma standard was run in ten replicates. Theresults of these assays were statistically analyzed with a mean value of0.10, a SD of 0.01, and a RSD of 9.3%. Thus, the LOD for the vitamin Cassay was 0.03 mg/dL.

Example 6 Assay Reportable Range and Linearity

To establish the linearity of vitamin C detection in the assay, threeseparate assays, each including one blank assigned as zero standard andeight spiked serum standards at concentrations ranging from 0.05 mg/dLto 10.00 mg/dL, were performed on separate days. A quadratic regressionfrom three consecutive runs yielded coefficient correlations of 0.9987or greater. A graph showing the linearity of the data is shown in FIG.2.

Example 7 Matrix Specificity

Matrix specificity was evaluated using human analyte-stripped anddelipidized serum (Cat. No. 1131-00, Biocell Labs, Carson, Calif. 90746,or equivalent), deionized (DI) water, 5% albumin, and in-house collectedpooled serum to determine whether patient samples could be diluted in alinear fashion. Two serum samples were spiked with high concentrationsof vitamin C: one at 25 mg/dL, and the other at 450 mg/dL. The spikedserums were then diluted from 2.5 to 500 times with the above matricesand analyzed. The study indicated that all four matrices could be usedfor dilution purposes up to 500 fold as long as the concentration afterdilution was about 0.5 mg/dL or higher. The results of this study arepresented in Table 4.

TABLE 4 Matrix Specificity of Serum/Plasma Vitamin C ConcentrationAlbumin 5% DI Water Biocell Serum In-house Serum Dilution (mg/dL)(mg/dL) (mg/dL) (mg/dL) (mg/dL) Day 1  0x 25.00    2.5x 10.00 8.27 9.598.76 8.15    2.5x 10.00 9.69 8.61 8.85 8.82  5x 5.00 4.59 4.34 4.44 4.32 5x 5.00 5.17 4.69 5.08 4.36  10x 2.50 2.19 2.36 2.16 2.37  10x 2.502.29 2.36 2.35 2.72  25x 1.00 0.86 0.83 0.88 0.88  25x 1.00 0.82 0.970.91 0.99  50x 0.50 0.43 0.24 0.36 0.42  50x 0.50 0.54 0.30 0.39 0.47100x 0.25 0.22 0.11 0.14 0.18 100x 0.25 0.26 0.09 0.19 0.22 Day 2  0x450.00  50x 9.00 8.89 8.87 8.34 8.74  50x 9.00 8.54 8.53 7.97 8.43 100x4.50 4.30 4.02 4.08 3.87 100x 4.50 4.56 4.32 3.97 4.33 200x 2.25 2.092.06 1.87 2.51 200x 2.25 1.97 1.91 2.09 2.20 500x 0.90 0.77 0.85 0.820.76 500x 0.90 0.96 0.71 0.80 0.75

Example 8 Recovery

A recovery study of vitamin C in spiked DI water and Biocell serumsamples was performed (in triplicate for concentrations of 0.10 mg/dL,0.39 mg/dL, 0.95 mg/dL, 2.70 mg/dL, 5.39 mg/dL, and 11.25 mg/dL). TheBiocell serum samples were subjected to the protein precipitationprocedure described in Example 2, above. The spiked DI water sampleswere injected without protein precipitation. Absolute recovery wascalculated by dividing the vitamin C concentration detected in the serumsamples by the vitamin C concentration detected in the DI water samples.The mean recoveries were 100.0%, 112.8%, 89.5%, 91.1%, 92.9%, and 85.8%respectively. All recoveries were acceptable, i.e., within the range of80% to 120%.

The contents of the articles, patents, and patent applications, and allother documents and electronically available information mentioned orcited herein, are hereby incorporated by reference in their entirety tothe same extent as if each individual publication was specifically andindividually indicated to be incorporated by reference. Applicantsreserve the right to physically incorporate into this application anyand all materials and information from any such articles, patents,patent applications, or other physical and electronic documents.

The methods illustratively described herein may suitably be practiced inthe absence of any element or elements, limitation or limitations, notspecifically disclosed herein. Thus, for example, the terms“comprising”, “including,” containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof. It is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the invention embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

The invention has been described broadly and generically herein. Each ofthe narrower species and subgeneric groupings falling within the genericdisclosure also form part of the methods. This includes the genericdescription of the methods with a proviso or negative limitationremoving any subject matter from the genus, regardless of whether or notthe excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, wherefeatures or aspects of the methods are described in terms of Markushgroups, those skilled in the art will recognize that the invention isalso thereby described in terms of any individual member or subgroup ofmembers of the Markush group.

That which is claimed is:
 1. A method for determining the amount ofvitamin C in a body fluid sample by tandem mass spectrometry, the methodcomprising: (a) purifying vitamin C from the body fluid sample by liquidchromatography; (b) generating a precursor ion of vitamin C having amass/charge ratio of 175.05±0.5; (c) generating one or more fragmentions of the precursor ion selected from the group of fragment ionshaving a mass/charge ratio of 114.85±0.5, and 86.85±0.5; and (d)detecting the amount of one or more of the ions generated in step (b) or(c) or both using tandem mass spectrometry and relating the determinedions to the amount of vitamin C in the body fluid sample.
 2. The methodof claim 1, wherein liquid chromatography comprises high performanceliquid chromatography (HPLC).
 3. The method of claim 1, wherein liquidchromatography comprises high turbulence liquid chromatography (HTLC).4. The method of claim 1, wherein the step of purifying vitamin C in abody fluid sample includes one or more purification steps prior toliquid chromatography.
 5. The method of claim 4, wherein saidpurification step comprises protein precipitation.
 6. The method ofclaim 1, wherein said body fluid sample is plasma or serum.
 7. Themethod of claim 1, wherein said ionizing comprises ionizing byatmospheric pressure chemical ionization (APCI).
 8. The method of claim1, further comprising adding a stabilizing agent to the test sampleprior to purifying the test sample.
 9. The method of claim 8, whereinsaid stabilizing agent is trichloroacetic acid (TCA).
 10. The method ofclaim 1, wherein said method has a lower limit of quantitation withinthe range of 10.0 mg/dL and 0.1 mg/dL, inclusive.
 11. The method ofclaim 1, wherein the amount of one or more vitamin C ions determined bymass spectrometry is related to the presence or amount of vitamin C inthe test sample by comparison to an internal standard.
 12. The method ofclaim 1, wherein the internal standard is ¹³C₆-L-ascorbic acid.
 13. Amethod for determining the amount of vitamin C in a body fluid sample bymass spectrometry, said method comprising: (a) contacting a body fluidsample with a stabilizing agent to inhibit vitamin C degradation; (b)purifying vitamin C from the sample by high performance liquidchromatography (HPLC); (c) generating a precursor ion of vitamin Chaving a mass/charge ratio of 175.05±0.5; (d) generating one or morefragment ions of the precursor ion selected from the group of fragmentions having a mass/charge ratio of 114.85±0.5, and 86.85±0.5; and (e)determining the amount of one or more vitamin C ions by massspectrometry, wherein the amount of the determined vitamin C ions isused to determine the amount of vitamin C in body fluid sample.
 14. Themethod of claim 13, further comprising subjecting the sample to apurification step prior to HPLC.
 15. The method of claim 14, whereinsaid purification step is high turbulence liquid chromatography (HTLC).16. The method of claim 14, wherein said purification step is proteinprecipitation.
 17. The method of claim 13, wherein said massspectrometry is tandem mass spectrometry.